Stat fax 2100 spectrophotometer

The 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based Cell Growth Determination kit (Sigma) was used for cell viability analyses. Cells were incubated for 24, 48, 72, or 168 h with test substances in 24-well plates in the differentiation medium: α-MEM, 10% FBS, 1% vol. ABAM, β-glycerophosphate 10 mM, ascorbic acid 50 µg/mL. Finally, cells were incubated for 4 h in a differentiation medium with the addition of MTT (0.5 mg/mL). Subsequently, the cell culture medium was removed, and the solvent was added (1 mM hydrochloric acid in isopropanol anhydride (Avantor)). Following gentle mixing, analysis was performed using the Stat-Fax 2100 spectrophotometer (Awareness Technology Inc., Palm City, FL, USA) at a wavelength of 630 nm (background absorbance was measured at 405 nm).
Caspase activity was analyzed by a Caspase-Glo 3/7 assay (Promega Corporation, Madison, WI, USA). Following cell stimulation, Caspase-Glo 3/7 Reagent was added (culture medium: Caspase Glo 3/7 reagent, 4:1) to cells in a 96-well plate. After incubation for 1 h, luminescence was measured in the TD20/20 luminometer (Turner Designs Inc., Sunnyvale, CA, USA).